The long range goal of studies in the principal investigator's laboratory is to understand, in as precise molecular detail as possible, the mechanism(s) by which alpha2 adrenergic receptors couple to distinct effector systems (such as inhibition of adenylate cyclase, suppression of calcium currents, activation of potassium currents) to elicit their diverse regulatory effects on excitation-secretion coupling. This next phase of the research is based on the discovery that a mutation in a highly conserved residue in the receptor, Asp79Asn, resulted in a loss of allosteric regulation of the receptor to sodium and, more significantly, a selective loss in coupling of the receptor to potassium currents in AtT20 cells. The selective loss of response by the mutated receptors offers an excellent opportunity to explore the roles of hormonal responsive currents in suppression of peptide hormone release in these cells and to gain insight into basic coupling mechanisms of the receptor.
Specific aims i nclude: 1) Determination of the relative abilities of wildtype and various mutant receptors to suppress peptide hormone secretion via measurement of ACTH release form AtT20 cells. 2) Evaluation of a series of mutants in the Asp79 position of the alpha2 receptor with regard to allosteric modulation by cations and coupling to inhibition of adenylate cyclase, ion channels, and secretion. 3) Clarification of the molecular basis for perturbation of coupling of the D79N mutation to activation of potassium currents in the AtT20 cells as a means to reveal the G protein involved in coupling this and other similar receptors to potassium currents. 4) Identification of alpha2A-adrenergic receptor associated effector complexes involved in receptor mediated signal transduction by selective immuno-isolation of effector proteins with wildtype versus functionally modified mutant receptors. Overall, the PI believes that these studies will provide new molecular insights regarding alpha2- adrenergic coupling to diverse signal transduction mechanisms as a first step toward developing novel therapeutic agents that are targeted downstream of receptor occupancy, and may provide a degree of specificity for function not attainable by simultaneous modulation of activity at all alpha2 receptors of a given pharmacological type.
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